Method for beam control in a scanning microscope, arrangement for beam control in a scanning microscope, and scanning microscope

ABSTRACT

A method and an arrangement for beam control in a scanning microscope are disclosed. The scanning microscope comprises means for acquiring and displaying ( 3 ) a preview image ( 7 ) and a microscope optical system ( 51 ). Means for marking ( 5 ) at least one region of interest ( 27, 29 ) in the preview image ( 7 ) are provided. A first beam deflection device ( 43, 67, 68 ) displaces the scan field ( 31, 33 ) onto the region of interest ( 27, 29 ); and a second beam deflection device ( 49, 72, 94 ) serves for meander-shaped scanning within the scan field ( 31, 33 ).

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority of the German patent application100 50 529.5 which is incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention concerns a method for scanning individual regionswith a scanning microscope, the regions of interest being distributedover the entire image field. It is possible to switch rapidly betweenthe individual regions of interest while maintaining the scanningmotion. The scanning motion can be accomplished by way of a suitablemotion of a scanning mirror.

[0003] The invention further concerns an arrangement for beam control inscanning microscopy.

[0004] In addition, the invention concerns a scanning microscope thatcomprises an arrangement for beam control which makes it possible toswitch rapidly between the individual regions of interest whilemaintaining the scanning motion. The scanning microscope can also beconfigured as a confocal scanning microscope. In particular, in thescanning microscope a light beam produced by an illumination system isguided over a specimen with the interposition of several optical means,and it contains at least one detector that, by way of the severaloptical means, detects a light proceeding from the specimen.

BACKGROUND OF THE INVENTION

[0005] In scanning microscopy, a sample is illuminated with a light beamin order to observe the reflected or fluorescent light emitted from thesample. The focus of the illuminating light beam is moved in a specimenplane by means of a controllable beam deflection device, generally bytilting two mirrors, the deflection axes usually being at right anglesto one another so that one mirror deflects in the X and the other in theY direction. The tilting of the mirrors is brought about, for example,using galvanometer positioning elements; both fast resonant galvanometerpositioning elements and slower (more accurate) non-resonant ones areused. In order to scan a sample in the specimen plane, it is importantthat the rotation axes of the mirror lie in or at least near a plane,also referred to as the Fourier plane, conjugated with the focal plane.One possible beam deflection device that meets the requirements fortelecentric imaging is known, for example, from DE 196 54 210. The powerlevel of the light coming from the specimen is measured as a function ofthe position of the scanning beam. Usually the positioning elements areequipped with sensors for ascertaining the present mirror position.

[0006] In confocal scanning microscopy specifically, a specimen isscanned in three dimensions with the focus of a light beam.

[0007] A confocal scanning microscope generally comprises a lightsource, a focusing optical system with which the light of the source isfocused onto a pinhole (called the excitation stop), a beam splitter, abeam deflection device for beam control, a microscope optical system, adetection stop, and the detectors for detecting the detected orfluorescent light. The illuminating light is coupled in via a beamsplitter. The fluorescent or reflected light coming from the specimenarrives via the beam deflection device back at the beam splitter, passesthrough it, and is then focused on the detection stop behind which thedetectors are located. Detected light that does not derive directly fromthe focus region takes a different light path and does not pass throughthe detection stop, thus yielding a point datum that results, bysequential scanning of the specimen, in a three-dimensional image. Athree-dimensional image is usually obtained by acquiring image data inlayers.

[0008] Ideally, the track of the scanning light beam on or in thespecimen describes a meander that fills the entire image field (scanningone line in the X direction at a constant Y position, then stopping theX scan and slewing by Y displacement to the next line to be scanned,then scanning that line in the negative X direction at constant Yposition, etc.). At high beam deflection speeds, deviations from theideal track occur because of the inertia of the deflecting moving parts,for example the galvanometer shaft and the mirrors. At usable scanningrates (>100 Hz) the scanning track of the light beam actually describesa sine curve, which in fact a necessitates a correction of thedeviations from the ideal situation resulting therefrom.

[0009] The power level of the light coming from the specimen is measuredat fixed time intervals during the scanning operation, and thus scannedone grid point at a time. The reading must be unequivocally associatedwith the pertinent scan position so that an image can be generated fromthe measured data. Advantageously this is done by also continuouslymeasuring the status data of the adjusting elements of the beamdeflection device, or (although this is less accurate) by directly usingthe reference control data of the beam deflection device.

[0010] In some microscopy applications the user is interested only ininformation about individual regions within the image field, while thesurrounding sample regions are not of interest. The regions of interestshould moreover be scanned as quickly as possible in succession.

[0011] Known arrangements offer only a limited capability for scanningindividual sample regions of interest. Scanning the entire image fieldand subsequently selecting the data of the regions of interest isfeasible, if at all, only to a limited extent given the required rapidsequential acquisition of information about the regions of interest.

[0012] The approach of sequentially scanning the individual regions ofinterest is better. It is possible in principle to activate the beamdeflection device in such a way that each of the regions of interest isseparately scanned, for example, in meander fashion, and the surroundingregions that are not of interest are not scanned. This procedure ispossible, however, only if the beam deflection device allows thescanning light beam to be specifically controlled and specificallydirected onto individual points in the image field.

[0013] This is not possible when using resonantly operating beamdeflection devices which are based, for example, on the use of resonantgalvanometers or micromirrors, because these beam deflection devicesoperate exclusively at the particular resonant frequency dictated bytheir design. It is not possible to “park” the light beam in one regionof the image field. Difficulties also occur with rapidly deflectingnon-resonantly operating beam deflection devices in terms of thepositionability that can be achieved, since the positioning elementsreact to an activation signal in delayed fashion because of theirinertia.

SUMMARY OF THE INVENTION

[0014] It is therefore the object of the invention to describe a methodfor scanning microscopic preparations with a light beam that solves theproblem described above.

[0015] This object is achieved by way of a method that comprises thefollowing steps:

[0016] acquiring a preview image;

[0017] marking at least one region of interest in the preview image;

[0018] displacing a scan field onto the region of interest by means of afirst beam deflection device; and

[0019] acquiring an image by meander-shaped scanning of the region ofinterest with a second beam deflection device.

[0020] What has been recognized according to the present invention isthat it is not necessary to forgo the use of fast or resonant beamdeflection devices if the scan field swept by the first beam deflectiondevice is displaced within the image field onto the regions of interestwith the aid of a further suitable beam deflection device that allowsexact positioning.

[0021] A further object of the invention is to create an arrangement forbeam control which makes it possible to switch rapidly among severalregions of interest and, in that context, to collect information fromregions of interest based on a consistent pattern.

[0022] This object is achieved by an arrangement for beam control in ascanning microscope. The arrangement comprises:

[0023] a scanning microscope defining a scan field;

[0024] means for acquiring and displaying a preview image

[0025] a microscope optical system;

[0026] means for marking at least one region of interest in the previewimage;

[0027] a first beam deflection device for displacing the scan field ontothe region of interest; and

[0028] a second beam deflection device for meander-shaped scanningwithin the scan field.

[0029] In a particular embodiment, according to the present invention animaging optical system is provided between the beam deflection devicesin order to guarantee the principle of telecentric scanning.

[0030] A further object of the invention is to create a scanningmicroscope that makes possible rapid sequential scanning of sampleregions of interest.

[0031] This object is achieved by a scanning microscope which comprises:

[0032] an arrangement for beam control,

[0033] means for acquiring and displaying a preview image

[0034] a microscope optical system,

[0035] means for marking at least one region of interest in the previewimage,

[0036] a first beam deflection device for displacing the scan field ontothe region of interest; and

[0037] a second beam deflection device for meander-shaped scanningwithin the scan field.

[0038] The invention has the advantage that it is not necessary to forgothe use of fast or resonant beam deflection devices if the scan fieldswept by the first beam deflection device is displaced within the imagefield onto the regions of interest with the aid of a second suitablebeam deflection device that allows exact positioning. Ideally, the trackof the scanning light beam on or in the specimen describes a rectangularcurve (scan one line in the X direction at a constant Y position, thenstop the X scan and slew by Y displacement to the next line to bescanned, then scan that line in the negative X direction at constant Yposition, etc.). At increasingly high deflection speeds, the scanningtrack deviates more and more from the rectangular shape. This phenomenonis attributable substantially to the inertia of the moving elements.With rapid scanning, the scanning track is more similar to a sine curve.In this context, the scan field is swept by the light beam in such a waythat the reversing points of the sinusoidal track lie outside the regionof interest. The scanning light beam thus describes approximatelystraight tracks on the region of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The subject matter of the invention is depicted schematically inthe drawings and will be described below with reference to the Figures,in which:

[0040]FIG. 1 schematically depicts selection of the regions of interest;

[0041]FIG. 2 schematically depicts the shape of the scanning track;

[0042]FIG. 3 shows a scanning microscope having the arrangementaccording to the present invention;

[0043]FIG. 4 shows a scanning microscope according to the presentinvention;

[0044]FIG. 5 shows a further embodiment of the scanning microscopeaccording to the present invention; and

[0045]FIG. 6 shows a further embodiment of the scanning microscopehaving an arrangement according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0046]FIG. 1 shows a PC 1 having a monitor 3 and a cursor controller 5.In one embodiment, cursor controller 5 is a computer mouse. Furtherpossibilities for a cursor controller 5 are, for example, a joystick, atrackball, or any other conceivable cursor control device. A previewimage 7 of the entire image field 19 is displayed on the monitor. Usingthe computer mouse, image areas 11, 13 of regions of interest 27, 29 aremarked with cursor 9. The marking is displayed in the form of borderinglines 15, 17.

[0047]FIG. 2 schematically depicts the shape of the scanning track,which is made up of partial scanning tracks 21, 23, and 25. Partialscanning tracks 21 and 25 are caused by a fast, resonant beam deflectiondevice, whereas partial scanning track 23 is caused by a beam deflectiondevice for specific positioning. Region of interest 27 is traversed bypartial scanning track 21, whereas region of interest 29 is traversed bypartial scanning track 25. Scan fields 31 and 33 are consequentlyscanned sequentially. Partial scanning track 23 is swept because of thedisplacement of scan fields 31, 33 using the second beam deflectiondevice for specific positioning.

[0048]FIG. 3 schematically shows a confocal scanning microscope. Lightbeam 37 coming from an illumination system 35 is reflected from a beamsplitter 39 via deflection mirror 41 to first beam deflection device 43for specific positioning, which contains a gimbal-suspended scanningmirror (not shown) and serves to displace the scan field in a planeperpendicular to the illumination direction. By way of an intermediateimage using an imaging optical system 47 that generates an intermediatefocal plane 45, light beam 37 arrives at second beam deflection device49 which is embodied as a K-mirror (see DE 196 54 210) and contains tworesonant galvanometers (not shown). Second beam deflection device 49provides meander-shaped scanning within the scan field. The size of ascan field can be varied by defining the deflection angle of thegalvanometers. Light beam 37 is consequently guided over or throughsample 53 via scanning lens 55, optical system 57 and through microscopeoptical system 51. In the case of nontransparent specimens 15, lightbeam 37 is guided over the specimen surface. In the case of biologicalsamples (preparations) or transparent samples, light beam 37 can also beguided through sample 53. This means that different focal planes ofsample 53 are successively scanned by the focus of light beam 37.Detected light 59 proceeding from sample 53 arrives, on the reverselight path via first and second beam deflection devices 43, 49, back atbeam splitter 39, passes through it, and is then detected with detector61. Detector 61 comprises a photomultiplier that converts the detectedlight data into electrical signals. Subsequent assembly of the signalsand allocation to the respective scan positions then yields athree-dimensional image of region of interest 27, 29 of sample 53.Illumination pinhole 63 and detection pinhole 65 that are usuallyprovided in a confocal scanning microscope are shown schematically forthe sake of completeness. Certain optical elements for guiding andshaping the light beams are omitted, however, for better clarity; theseare sufficiently known to those skilled in this art.

[0049]FIG. 4 shows a confocal scanning microscope having a first beamdeflection device 67 for specific position. Second beam deflectiondevice 72, which is constituted by a first and a second beam deflectionmodule 71 and 75, is provided for rapid scanning of the scan field. Thetwo beam deflection modules 71 and 75 can, for example, each contain aresonantly oscillating micromirror. Imaging optical systems 69, 73 areprovided between first and second beam deflection devices 67 and firstand second beam deflection modules 71 and 75 to maintain the telecentricprinciple.

[0050]FIG. 5 shows a confocal scanning microscope having a total of fourbeam deflection modules 77, 79, 81, 83 that are distributed respectivelyamong two beam deflection devices 78 and 82. Beam deflection modules 77and 81 deflect in the X direction, whereas beam deflection modules 79and 83 deflect in the Y direction. Beam deflection device 78 serves toposition scan field 31, 33. It contains, for example, galvanometers thatoperate non-resonantly. Beam deflection device 82 operates resonantly,and serves to scan scan field 31, 33 in meander-shaped fashion. Beamdeflection modules 77, 79 and 81, 83 are arranged close to planes 89 and87, respectively, that are conjugated with the focal plane of microscopeoptical system 51. Imaging optical system 85 is provided to maintain thetelecentric principle.

[0051]FIG. 6 shows a further exemplary embodiment of a confocal scanningmicroscope. Here it is the gimbal-mounted mirror 91, driven innon-resonant fashion, that serves to position scan field 31, 33. Inorder to maintain the telecentric scanning principle, mirror 93 thatdeflects in the Y direction and is driven by a resonant galvanometer,and mirror 95 that deflects in the X direction and is driven by aresonant galvanometer, are arranged close to plane 99 conjugated withthe focal plane of microscope optical system 51. Imaging optical system98, which is made up of optical systems 97 and 101 and generates at therotation point of mirror 91 a further plane 99 conjugated with the focalplane of microscope optical system 51, is located between mirrors 93 and91. In this exemplary embodiment, second beam deflection device 94comprises mirrors 93 and 95.

[0052] First beam deflection device 43, 67, 78 and/or second beamdeflection device 49, 72, 82, 94 contain, inter alia, a gimbal-mountedmirror or a micromirror or an acoustooptical deflector or a galvanometermirror or a resonantly oscillating mirror system.

[0053] The present invention was described with reference to aparticular exemplary embodiment. It is, however, self-evident thatchanges and modifications can be made without thereby leaving the rangeof protection of the claims recited hereinafter.

PARTS LIST

[0054] 1 PC

[0055] 3 Monitor

[0056] 5 Cursor controller

[0057] 7 Preview image

[0058] 9 Cursor

[0059] 11 Image area

[0060] 13 Image area

[0061] 15 Bordering line

[0062] 17 Bordering line

[0063] 19 Image field

[0064] 21 Partial scanning track

[0065] 23 Partial scanning track

[0066] 25 Partial scanning track

[0067] 27 Region of interest

[0068] 29 Region of interest

[0069] 31 Scan field

[0070] 33 Scan field

[0071] 35 Illumination system

[0072] 37 Light beam

[0073] 39 Beam splitter

[0074] 41 Deflection mirror

[0075] 43 First beam deflection device

[0076] 45 Intermediate focal plane

[0077] 47 Imaging optical system

[0078] 49 Second beam deflection device

[0079] 51 Microscope optical system

[0080] 53 Sample

[0081] 55 Scanning lens

[0082] 57 Optical system

[0083] 59 Detected light

[0084] 61 Detector

[0085] 63 Illumination pinhole

[0086] 65 Detection pinhole

[0087] 67 First beam deflection device

[0088] 69 Imaging optical system

[0089] 71 First beam deflection module

[0090] 72 Second beam deflection device

[0091] 73 Imaging optical system

[0092] 75 Second beam deflection module

[0093] 77 First beam deflection module

[0094] 78 First beam deflection device

[0095] 79 Second beam deflection module

[0096] 81 First beam deflection module

[0097] 82 Second beam deflection device

[0098] 83 Second beam deflection module

[0099] 85 Imaging optical system

[0100] 87 Conjugated plane

[0101] 89 Conjugated plane

[0102] 91 Mirror

[0103] 93 Mirror

[0104] 94 Second beam deflection device

[0105] 95 Mirror

[0106] 97 Optical system

[0107] 98 Imaging optical system

[0108] 99 Conjugated plane

[0109] 101 Optical system

What is claimed is:
 1. A method for beam control in a scanningmicroscope, comprising the following steps: acquiring a preview image;marking at least one region of interest in the preview image; displacinga scan field onto the region of interest by means of a first beamdeflection device; and acquiring an image by meander-shaped scanning ofthe region of interest with a second beam deflection device.
 2. Themethod as defined in claim 1, comprising a further step in which thefirst beam deflection device transfers the meander-shaped scanning of aregion of interest into the meander-shaped scanning of a further regionof interest.
 3. The method as defined in claim 2, wherein themeander-shaped scanning of the regions of interest proceeds inaccordance with a defined sequence and thereby acquires information fromthe regions of interest.
 4. The method as defined in claim 3, whereinthe sequence of the meander-shaped scanning of the regions of interestis user-defined.
 5. The method as defined in claim 3, wherein thesequence of the meander-shaped scanning of the regions of interest isperformed automatically on the basis of the information obtained fromthe regions of interest.
 6. An arrangement for beam control comprising:a scanning microscope defining a scan field; means for acquiring anddisplaying a preview image a microscope optical system; means formarking at least one region of interest in the preview image; a firstbeam deflection device for displacing the scan field onto the region ofinterest; and a second beam deflection device for meander-shapedscanning within the scan field.
 7. The arrangement for beam control asdefined in claim 6, wherein the first beam deflection device transfersthe meander-shaped scanning of a region of interest into themeander-shaped scanning of a further region of interest.
 8. Thearrangement as defined in claim 6, wherein the second beam deflectiondevice is configured with two beam deflection modules.
 9. Thearrangement as defined in claim 6, wherein the first beam deflectiondevice is configured with two beam deflection modules.
 10. Thearrangement as defined in claim 6, wherein the first beam deflectiondevice and second beam deflection device consists essentially of agimbal-mounted mirrors, a micromirror, an acoustooptical deflector, agalvanometer mirror or a resonantly oscillating mirror system.
 11. Thearrangement as defined in claim 6, wherein the first beam deflectiondevice is arranged substantially in the vicinity of a plane conjugatedwith the focal plane of the microscope optical system.
 12. Thearrangement as defined in claim 6, wherein the second beam deflectiondevice is arranged substantially in the vicinity of a plane conjugatedwith the focal plane of the microscope optical system.
 13. Thearrangement as defined in claim 11, wherein an imaging optical system isprovided between the first beam deflection device and second beamdeflection device.
 14. The arrangement as defined in claim 13, wherein arelay optical system is provided between the first beam deflectiondevice and second beam deflection device.
 15. A scanning microscopecomprising: an arrangement for beam control, means for acquiring anddisplaying a preview image a microscope optical system, means formarking at least one region of interest in the preview image, a firstbeam deflection device for displacing the scan field onto the region ofinterest; and a second beam deflection device for meander-shapedscanning within the scan field.
 16. The scanning microscope as definedin claim 15, wherein the first beam deflection device transfers themeander-shaped scanning of one region of interest into themeander-shaped scanning of a further region of interest.
 17. Thescanning microscope as defined in claim 15, wherein the second beamdeflection device is configured with two beam deflection modules. 18.The scanning microscope as defined in claim 15, wherein the first beamdeflection device is configured with two beam deflection modules. 19.The scanning microscope as defined in claim 15, wherein the first beamdeflection device and second beam deflection device consists essentiallyof gimbal-mounted mirrors, a micromirror, an acoustooptical deflector, agalvanometer mirror or a resonantly oscillating mirror system.
 20. Thescanning microscope as defined in claim 15, wherein the first beamdeflection device is arranged substantially in the vicinity of a planeconjugated with the focal plane of the microscope optical system. 21.The scanning microscope as defined in claim 15, wherein the second beamdeflection device is arranged substantially in the vicinity of a planeconjugated with the focal plane of the microscope optical system. 22.The scanning microscope as defined in claim 20, wherein an imagingoptical system is provided between the first beam deflection device andsecond beam deflection device.
 23. The scanning microscope as defined inclaim 22, wherein a relay optical system is provided between the firstbeam deflection device and second beam deflection device.